1. Technical Field
The invention relates to the field of biomedical sciences, and in particular relates to novel advanced glycation end product (“AGE”), inhibitors methods for suppression of expression of a gene encoding the receptor for advanced glycation end products (RAGE), and suppression of pro-inflammatory signals and genes triggered by RAGE including NF-kB, MCP-1, TNF-a, NADPH-oxidase.
2. Description of the Background Art
Advanced glycation end products (AGEs), the products of non-enzymatic glycation and oxidation of proteins and lipids, accumulate in diverse biological settings, such as diabetes, inflammation, renal failure and aging. Ahmed N, Diabetes Res Clin Pract. 67: 3-21, 2005; Vlassara H, J Intern Med. 251: 87-101, 2001. AGEs have been proposed to play a crucial role in the pathogenesis of diabetic vascular complications and atherogenesis in non-diabetic subjects. Rojas A, Morales M A., Life Sci. 76: 715-730, 2004; Yamagishi S, Imaizumi T, Curr Pharm Des 11: 2279-2299, 2005. Patients with diabetes exhibit an increased propensity to develop atheroschlerosis, with its sequelae acute myocardial infarction and stroke. Basta G, Schmidt A M, De Caterina R, Cardiovasc Res. 63: 582-592, 2004; Jakus V, Rietbrock N. Physiol Res. 53:1131-142, 2004.
Non-enzymatic glycation (also known as the Maillard reaction) is a complex series of reactions between reducing sugars and the amino groups of proteins, lipids, and DNA which leads to browning, fluorescence and cross-linking. Bucala et al., Proc. Natl. Acad. Sci. USA 90:6434-6438, 1993; Bucala et al., Proc. Natl. Acad Sci. USA 81:105-109, 1984; Singh et al., Diabetologia 44:129-146, 2001. This complex cascade of condensations, rearrangements and oxidation produces heterogeneous, irreversible, proteolysis-resistant, antigenic products known as advanced glycation end products. Singh et al., Diabetologica 44:129-146, 2001; Ulrich and Cerami, Rec. Prog. Hormone Res. 56:1-2, 2001. Examples of these AGEs are Nε-(carboxymethyl) lysine (CML), Nε-(carboxyethyl) lysine (CEL), Nε-(carboxymethyl)cysteine (CMC), arg-pyrimidine, pentosidine and the imidazolium crosslinks methyl-gloxal-lysine dimer (MOLD) and glyoxal-lysine dimer (GOLD). Thorpe and Baynes, Amino Acids 25:275-281, 2002; Chellan and Nagaraj, Arch. Biochem. Biophys. 368:98-104, 1999. This type of glycation begins with the reversible formation of a Schiff's base, which undergoes a rearrangement to form a stable Amadori product.
Both Schiff's bases and Amadori products further undergo a series of reactions through dicarbonyl intermediates to form AGEs. Lipid peroxidation of polyunsaturated fatty acids (PUFA), such as arachidonic acid and linoleic acid, also yield carbonyl compounds. Some of these compounds are identical to those formed from carbohydrates, such as MG and GO, and others are characteristic of lipid, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE), and 2-hydroxyheptanal (2HH). See Baynes and Thorpe, Free Rad. Biol. Med. 28:1708-1716, 2000; Fu et al., J. Biol. Chem. 271:9982-9986, 1996; Miyata et al., FEBS Lett. 437:24-28, 1998; Miyata et al., J. Am. Soc. Nephrol. 11:1744-1752, 2000; Requena et al., Nephrol. Dial. Transplant. 11 (supp. 5):48-53, 1996; Esterbauer et al., Free Radic. Biol. Med. 11:81-128, 1991; Requena et al., J. Biol. Chem. 272:17473-14779, 1997; Slatter et al., Diabetologia 43:550-557, 2000. These reactive carbonyl species (RCSs) rapidly react with lysine and arginine residues of proteins, resulting in the formation of advanced lipoxidation end products (ALEs) such as Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL), GOLD, MOLD, malondialdehyde-lysine (MDA-lysine), 4-hydroxynonenal-lysine (4-HNE-lysine), hexanoyl-lysine (Hex-lysine), and 2-hydroxyheptanoyl-lysine (2HH-lysine). Thorpe and Baynes, Amino Acids 25:275-281, 2002; Miyata et al., FEBS Lett. 437:24-28, 1998; Miyata et al., J. Am. Soc. Nephrol. 11:1744-1752, 2000; Uchida et al., Arch. Biochem. Biophys. 346:45-52, 1997; Baynes and Thorpe, Free Rad. Biol. Med. 28:1708-1716, 2000. Since CML, CEL, GOLD and MOLD can result from lipid and carbohydrate metabolism, these chemical modifications on tissue proteins can serve as biomarkers of oxidative stress resulting from sugar and lipid oxidation. Fu et al., J. Biol. Chem. 271:9982-9986, 1996; Requena et al., Nephrol. Dial. Transplant. 11 (supp. 5):48-53, 1996.
In human diabetic patients and in animal models of diabetes, these non-enzymatic reactions are accelerated and cause accumulation of AGEs on long-lived structural proteins such as collagen, fibronectin, tubulin, lens crytallin, myelin, laminin and actin, in addition to hemoglobin, albumin, LDL-associated proteins and apoprotein. The structural and functional integrity of the affected molecules, which often have major roles in cellular functions, are perturbed by such modifications, with severe consequences on organs such as kidney, eye, nerve, and micro-vascular functions, which consequently leads to various diabetic complications such as nephropathy, atherosclerosis, microangiopathy, neuropathy and retinopathy. Boel et al., J. Diabetes Complications 9:104-129, 1995; Hendrick et al., Diabetologia 43:312-320, 2000; Vlassara and Palace, J. Intern. Med. 251:87-101, 2002.
Research results indicate that reactive carbonyl species such as MGO, GO, GLA, dehydroascorbate, 3-deoxyglucosone and malondialdehyde, are potent precursors of AGE/ALE formation and protein crosslinking. Lyons and Jenkins, Diabetes Rev. 5:365-391, 1997; Baynes and Thorpe, Diabetes 48:1-9, 1999; Miyata et al., J. Am. Soc. Nephrol. 11:1744-1752, 2000; Thornalley st al., Biochem. J. 344:109-116, 1999. In vitro studies further suggest that these carbonyl originate mainly from ascorbate and polyunsaturated fatty acids and not from glucose per se. Miyata et al., FEBS Lett. 437:24-28, 1993.
Direct evidence implicates the contribution of AGEs/ALEs in the progression of diabetic complications in different lesions of the kidneys, the rat lens, and in atherosclerosis. Horie et al., J. Clin. Invest. 100:2995-3004, 1997; Matsumoto et al., Biochem. Biophys. Res. Commun. 241:352-354, 1997; Bucala and Vlassara, Exper. Physiol. 82:327-337, 1997; Bucala and Rahbar, in: Endocrinology of Cardiovascular Function. E. R. Levin and J. L. Nadler (eds.), 1998. Kluwer Acad. Publishers, pp. 159-180; Horie et al., J. Clin. Invest. 100:2995-3004, 1997; Friedman, Nephrol. Dial. Transplant. 14 (supp. 3):1-9, 1999; Kushiro et al., Nephron 79:458-468, 1998. Several lines of evidence indicate that hyperglycemia in diabetes causes the increase in reactive carbonyl species (RCS) such as methylglyoxal, glycolaldehyde, glyoxal, 3-deoxyglucosone, malondialdehyde, and hydroxynonenal. “Carbonyl stress” leads to increased modification of proteins and lipids, through reactive carbonyl intermediates forming adducts with lysine residues of proteins, followed by oxidative stress and tissue damage. Lyons and Jenkins, Diabetes Rev. 5:365-391, 1997; Baynes and Thorpe, Diabetes 48:1-9, 1999; Miyata et al., J. Am. Soc. Nephrol. 11:1744-1752, 2000.
Through generation of reactive oxygen species (ROS), reactive carbonyl species (RCS) and reactive nitrogen species (RNS), AGEs contribute to tissue injury by alteration of extracellular matrix structures through formation of protein crosslinks, and alteration of intracellular short-lived proteins such as metabolic enzymes and mitochondrial protein complexes. DeGroot J. Curr Opin Pharmacol. 4: 301-305, 2004; Rosca M G, Mustata T G, Kinter M T, et al., Am J Physiol Renal Physiol. 289: F420-F430, 2005. Multiple receptor independent and receptor dependent pathways linking AGEs/ALEs to cellular and tissue dysfunction have been proposed. Schmidt A M, Hori O, Brett J, et al. Arterioscler Thromb 14: 1521-1528, 1994; Vlassara H., Diabetes Metab Res Rev. 17: 436-443, 2001. Modulation of cellular functions through interactions with specific cell surface receptors, the best characterized of which is the receptor of AGE (RAGE), has been extensively explored. Schmidt A M, Yan S D, Yan S F, Stern D M., Biochim. Biophys. 1498:99-111, 2000; Kim W, Hudson B, Moser B, et al., Ann N Y Acad Sci. 1043: 553-561, 2005; Bucciarelli L G, Wendt T, Rong L, et al., Cell Mol Life Sci. 59: 1117-1128, 2002.
Binding of AGEs to RAGE activates intracellular signaling processes, thus mediating pro-inflammatory AGE effects. Basta G, Lazzerini G, Massaro M, et al., Circulation 105: 816-822, 2002; Chavakis T, Bierhaus A, Nawroth P P, Microbes Infect. 6: 1219-1225, 2004. Previous work has demonstrated that RAGEs are present at low levels on the surface of vascular cells, smooth muscle cells, fibroblasts, and monocyte/macrophages. Bucciarelli L G, Wendt T, Rong L, et al., Cell Mol Life Sci. 59: 1117-1128, 2002.
In endothelial cells, tumor necrosis factor-α (TNF-α) as well as AGEs themselves, upregulate RAGE expression, thus rendering these cells more susceptible to pro-inflammatory AGE effects Basta G, Schmidt A M, De Caterina R., Cardiovasc Res. 63: 582-592, 2004. In addition to AGEs, peptides like S100/calgranulins, β-amyloid, and amphoterin have been shown to activate RAGE. Liliensiek B, Weigand M A, Bierhaus A, et al., J Clin Invest. 113: 1641-1650, 2004. In endothelial cells, binding of RAGEs to these ligands activates the transcription factor nuclear factor-κb (NF-κb), subsequently leading to increased expression of pro-atherogenic mediators such as monocyte chemoattractant protein-1 (MCP-1) or vascular cell adhesion molecule-1 (VCAM-1) Basta G, Lazzerini G, Massaro M, et al., Circulation 105: 816-822, 2002; Wautier J L, Schmidt A M. 2004. Circ Res. 95:233-238, 2004.
AGE formation has been proposed to be the key step in creating a nidus for the amplification of stress pathways and is hypothesized to be involved in a vicious cycle of AGE→inflammation→ROS→AGE→ more inflammation. Ramasamy R, Vannucci S J, Yan S S, et al., Glycobiology. 15:16R-28R, 2005. In vitro experiments as well as animal data suggest that limiting RAGE expression in vascular cells might be an intriguing concept to modulate atherogenesis and inflammatory disorders Hudson B I, Schmidt A M., Pharm Res. 21: 1079-1086, 2005. Suppression of enhanced expression of endothelial RAGE has been achieved by using extracellular domain of RAGE (sRAGE) Park L, et al., Nat. Med. 4: 1025-1031, 2001; Bucciarelli L G, Wendt T, Qu W, et al., Circulation 106: 2827-2835, 2001, anti RAGE IgG antibody (Rong L L, Trojaborg W, Qu W, et al., FASEB J. 18:11812-11817, 2004) and by thiazolidinediones like pioglitazone and roziglitazone. Marx N, Walcher D, Ivanova N, et al., Diabetes 53: 2662-2668, 2004.
Over the years, several natural and synthetic compounds have been proposed and advanced as potential AGE/ALE inhibitors. These include aminoguanidine, pyridoxamine, OPB-9195, carnosine, metformin, as well as some angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II type 1 receptor blockers (ARB), derivatives of aryl (and heterocyclic) ureido, and aryl (and heterocyclic) carboxamido phenoxyisobutyric acids. Rahbar et al., Biochem. Biophys. Res. Commun. 262:651-656, 1999; Rahbar et al., Mol. Cell. Biol. Res. Commun. 3:360-366, 2000; Rahbar and Figarola, Curr. Med. Chem. (Immunol. Endocr. Metabol. Agents) 2:135-161, 2002; Rahbar and Figarola, Curr. Med. Chem. (Immunol. Endocrin. Metabol.) 2:174-186, 2002; Forbes et al., Diabetes 51:3274-3282, 2002; Metz et al., Arch. Biochem. Biophys. 419:41-49; Nangaku et al., J. Am. Soc. Nephrol. 14:1212-1222, 2003; Rahbar and Figarola, Arch. Biochem. Biophys. 419:63-79, 2003. Recently, some of these compounds were found to be effective AGE inhibitors in vivo and to prevent the development of diabetic nephropathy in a streptozotocin-induced diabetes.
Over the last decade, evidence has accumulated implicating AGEs/ALEs as major factors in the pathogenesis of diabetic nephropathy and other complications of diabetes. Administration of AGEs to non-diabetic rats leads to glomerulosclerosis and albuminuria, indicating that AGEs alone may be sufficient to cause renal injury in diabetes. Vlassara et al., Proc. Natl. Acad. Sci. USA 91:11704-11708, 1994. Diabetic animals fed with a diet low in glycoxidation products developed minimal symptoms of diabetic nephropathy compared with animals fed with diet high in glycoxidation products. Zheng et al., Diabetes Metab. Res. Rev. 18:224-237, 2002. It is widely accepted that AGEs/ALEs contribute to diabetic tissue injury by at least two major mechanisms. Browlee, Nature 414:813-820, 2001; Stith et al., Expert Opin. Invest. Drugs 11:1205-1223, 2002; Vlassara and Palace, J. Intern. Med. 251:87-101, 2002. The first is receptor-independent alterations of the extracellular matrix architecture and function of intracellular proteins by AGE/ALE formation and AGE/ALE-protein crosslinking. The other is receptor-dependent modulation of cellular functions through interaction of AGE with various cell surface receptors, especially RAGE. Wendt et al., Am. J. Pathol. 162:1123-1137, 2003; Vlassara, Diabetes Metab. Res. Rev. 17:436-443, 2001; Kislinger et al., J. Biol. Chem. 274:31740-3174, 1999.
Advanced glycation/lipoxidation end products (AGEs/ALEs) also have been implicated in the pathogenesis of a variety of debilitating diseases such as atherosclerosis, Alzheimer's and rheumatoid arthritis, as well as the normal aging process. The pathogenic process is accelerated when elevated concentrations of reducing sugars or lipid peroxidation products are present in the blood and in the intracellular environment such as occurs with diabetes. Both the structural and functional integrity of the affected molecules become perturbed by these modifications and can result in severe consequences in the short and long term. Because hyperlipidemia, hyperglycemia, diabetes and syndromes such as “metabolic syndrome” are common and are a common cause of morbidity and mortality, methods to counteract the symptoms and consequences of these metabolic states are needed in the art.
New classes of compounds as inhibitors of AGE formation and protein crosslinking have been reported previously. Rahbar S, Figarola J L., Arch Biochem Biophys. 419: 63-79, 2003. More recently, several of these LR compounds were found effective in preventing the development of diabetic nephropathy in STZ-induced diabetic animals Figarola J L, Scott S, Loera S, et al., Diabetologia 46: 1140-1152m 2005; Figarola J L, Scott S, Loera S, et al., Diabetes Metab Res Rev. 21: 533-544, 2005. To date, methylene bis (4,4′-(2-chlorophenylureidophenoxy-isobutyric acid) (referred to herein as “LR-90”) has been found to be the most powerful among all other compounds in the LR-series.